Therapeutic antibodies and uses thereof

ABSTRACT

Provided herein are methods, compositions, and uses relating to inhibitors of stem cell factor. For example, provided herein are antibodies targeting stem cell factor and methods for treating fibrotic and tissue remodeling diseases.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 61/844,728, filed Jul. 10, 2013, the disclosure ofwhich is herein incorporated by reference in its entirety.

FIELD OF INVENTION

Provided herein are methods, compositions, and uses relating to antibodyinhibitors of stem cell factor. For example, provided herein areantibodies targeting stem cell factor and methods for treating fibroticand tissue remodeling diseases.

BACKGROUND

Diseases involving tissue remodeling and fibrosis are a leading cause ofdeath worldwide. Nearly 45 percent of all natural deaths in the westernworld are attributable to some type of chronic fibroproliferativedisease and the associated health care costs are in the billions ofdollars. Tissue remodeling is the reorganization or renovation ofexisting tissues, which can either change the characteristics of atissue (e.g., blood vessel remodeling) or participate in establishingthe dynamic equilibrium of a tissue (e.g., bone remodeling). Fibrosis isthe formation or development of excess fibrous connective tissue in anorgan or tissue as a reparative or reactive process, as opposed toformation of fibrous tissue as a normal constituent of an organ ortissue. Fibrosis affects nearly all tissues and organ systems, andfibrotic tissue remodeling can influence cancer metastasis andaccelerate chronic graft rejection in transplant recipients. Diseases inwhich fibrosis is a major cause of morbidity and mortality include theinterstitial lung diseases, liver cirrhosis, kidney disease, heartdisease, and systemic sclerosis, among others.

Stem cell factor (SCF) and its receptor c-Kit have been implicated infibrotic and tissue remodeling diseases (El-Koraie, et al., Kidney Int.60: 167 (2001); Powell, et al., Am. J. Physiol. 289: G2 (2005); ElKossi, et al., Am. J. Kidney Dis. 41: 785 (2003); Powell, et al., Am. J.Physiol. 277: C183 (1999)). c-Kit is a type III receptor-tyrosine kinasethat is present in many cell types (Orr-Urtreger et al., Development109: 911 (1990)). It is also expressed in the early stages ofdifferentiation (Andre et al., Oncogene 4: 1047 (1989)) and certaintumors exhibit elevated expression of c-kit. SCF is a ligand specificfor the c-Kit receptor kinase. Binding causes dimerization of c-Kit andactivation of its kinase activity. SCF was first isolated from thesupernatant of murine fibroblasts. At the time, SCF was called mast cellgrowth factor (MGF) (Williams et al., Cell 63: 167 (1990)) orhematopoietic growth factor KL (Kit ligand) (Huang et al., Cell 63: 225(1990)). A homologue was subsequently isolated from rat liver cells anddesignated stem cell factor (SCF) (Zsebo et al., Cell 63: 195 (1990)).The corresponding human protein is designated variously as SCF, MGF, orSteel Factor (SF) (Cell 63: 203 (1990)).

Previous studies have suggested that an inhibitor of c-Kit receptortyrosine kinase can significantly inhibit aberrant tissue fibrosis (see,e.g., Aono, Am. J. Respir. Crit. Care Med. 171: 1279 (2005); Vuorinen,et al., Exp. Lung Res. 33: 357 (2007); Vittal, et al., J. Pharmacol.Exp. Ther. 321:35 (2007); Distler, et al., Arthritis Rheum 56: 311(2007)). However, this inhibitor has several disadvantages. It needs tobe given systemically by oral administration, it has some toxicityassociated with its use, and the compound must be deliveredintracellularly for efficacy. Consequently, alternative therapies areneeded.

SUMMARY

Provided herein are methods, compositions, and uses relating toinhibitors of stem cell factor. For example, provided herein areantibodies targeting stem cell factor and methods for treating fibroticand tissue remodeling diseases as well as for research and diagnosticuses.

Embodiments of the present invention provide an isolated recombinantmonoclonal anti-stem cell factor (SCF) antibody comprising: (a) a lightchain variable domain comprising the amino acid sequence of SEQ ID NO:4or sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or fractionsthereof) identity to SEQ ID NO:4; and (b) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:2 or sequences with atleast 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or fractions thereof identity toSEQ ID NO:2. In some embodiments, the light chain variable region hasthe amino acid sequence of SEQ ID NO:4 and the heavy chain variableregion has the amino acid sequence of SEQ ID NO:2. In some embodiment,the antibody is monovalent and comprises an Fc region. In someembodiments, the antibody is bivalent. In some embodiments, the antibodyis bispecific. In some embodiments, the antibody is an antibody fragmentselected from, for example a Fab, a Fab′-SH, an Fv, an scFv, or a(Fab′)₂ fragment. In some embodiments, the antibody comprises a singleFab region linked to an Fc region. Antibodies of the invention canfurther comprise any suitable framework and/or light chain variabledomain sequences, provided SCF binding activity is substantiallyretained. For example, in some embodiments, these antibodies furthercomprise a human subgroup III heavy chain framework consensus sequence.In one embodiment, these antibodies further comprise a human κI lightchain framework consensus sequence.

Further embodiments provide a pharmaceutical composition comprising anyof the aforementioned antibodies and a pharmaceutically acceptablecarrier.

Additional embodiments provide a nucleic acid encoding any of theaforementioned antibodies. In some embodiments, the nucleic acidcomprises a nucleic acid encoding a light chain variable regioncomprising SEQ ID NO:3 or sequences that are at least 80% (e.g., 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or fractions thereof or alternative codons thatencode the amino acids of SEQ ID NO:4) homologous to SEQ ID NO:3 and anucleic acid encoding a heavy chain variable region comprising SEQ IDNO:1 or sequences that are at least 80% (e.g., 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orfractions thereof or alternative codons that encode the amino acids ofSEQ ID NO:2) homologous to SEQ ID NO:1. In some embodiments, the nucleicacid encoding a light chain variable region comprises SEQ ID NO:3 andthe nucleic acid encoding a heavy chain variable region comprises SEQ IDNO:1.

In some embodiments, the present invention provides the use of theaforementioned pharmaceutical composition or antibodies in the treatmentof a lung, fibrotic or tissue remodeling disease.

In some embodiments, the present invention provides a method of treatinga fibrotic or tissue remodeling disease comprising administering theaforementioned pharmaceutical composition or antibodies to a subjectwith or at risk for a fibrotic or tissue remodeling disease. In someembodiments, the subject has an abnormal activity of stem cell factor orabnormal collagen production. In some embodiments, the disease isfibrosis, a remodeling disease, or a pulmonary disease (e.g., idiopathicpulmonary fibrosis, chronic obstructive pulmonary disease, acuterespiratory distress syndrome, cystic fibrosis, peribronchial fibrosis,hypersensitivity pneumonitis, asthma, pulmonary arterial hypertension(PAH), sclerodoma, inflammation, liver cirrhosis, renal fibrosis,parenchymal fibrosis, endomyocardial fibrosis, mediatinal fibrosis,nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax,hepatic fibrosis, fibromyalgia, gingival fibrosis, or radiation-inducedfibrosis). In some embodiments, the pharmaceutical composition isdelivered into an airway of the subject by e.g., intranasal orinhalational (e.g. dry powder or nebulizer) administration. In someembodiments, the administering reduces an activity of a receptor and/orreduces an interaction of stem cell factor with a receptor (e.g., areceptor tyrosine kinase such as

c-Kit). In some embodiments, the administering results in a directinhibition of fibroblast activation. In some embodiments, theadministering results in inhibition of progression of signs or symptomsof a disease.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary CDR and Framework regions of the antibodies ofembodiments of the present disclosure.

DETAILED DESCRIPTION

Provided herein are methods, compositions, and uses relating toinhibitors of stem cell factor. For example, provided herein areantibodies targeting stem cell factor, methods of producing antibodiestargeting stem cell factor, and methods for treating fibrotic and tissueremodeling diseases as well as for research and diagnostic uses. In someembodiments, the compositions, methods, and uses herein providetherapies relating to inhibiting stem cell factor (SCF). Someembodiments provide an isolated antibody that targets SCF. In someembodiments, inhibiting SCF affects the activity of c-Kit. Thecompositions, methods, and uses provided herein find use in treatingfibrotic diseases and maladies associated with tissue remodeling.

DEFINITIONS

To facilitate an understanding of embodiments of the present technology,a number of terms and phrases are defined below. Additional definitionsare set forth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably. A“protein” or “polypeptide” encoded by a gene is not limited to the aminoacid sequence encoded by the gene, but includes post-translationalmodifications of the protein.

Where the term “amino acid sequence” is recited herein to refer to anamino acid sequence of a protein molecule, “amino acid sequence” andlike terms, such as “polypeptide” or “protein” are not meant to limitthe amino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule. Furthermore, an “aminoacid sequence” can be deduced from the nucleic acid sequence encodingthe protein.

The term “nascent” when used in reference to a protein refers to a newlysynthesized protein, which has not been subject to post-translationalmodifications, which includes but is not limited to glycosylation andpolypeptide shortening. The term “mature” when used in reference to aprotein refers to a protein which has been subject to post-translationalprocessing and/or which is in a cellular location (such as within amembrane or a multi-molecular complex) from which it can perform aparticular function which it could not if it were not in the location.

The term “portion” when used in reference to a protein (as in “a portionof a given protein”) refers to fragments of that protein. The fragmentsmay range in size from four amino acid residues to the entire aminosequence minus one amino acid (for example, the range in size includes4, 5, 6, 7, 8, 9, 10, or 11 . . . amino acids up to the entire aminoacid sequence minus one amino acid).

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions(i.e., additions), or both. Guidance in determining which and how manyamino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

The term “domain” when used in reference to a polypeptide refers to asubsection of the polypeptide which possesses a unique structural and/orfunctional characteristic; typically, this characteristic is similaracross diverse polypeptides. The subsection typically comprisescontiguous amino acids, although it may also comprise amino acids whichact in concert or which are in close proximity due to folding or otherconfigurations. Examples of a protein domain include the transmembranedomains, and the glycosylation sites.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide. Thus, “a nucleotide comprising at least aportion of a gene” may comprise fragments of the gene or the entiregene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

The terms “an oligonucleotide having a nucleotide sequence encoding agene” or “a nucleic acid sequence encoding” a specified polypeptiderefer to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNAor RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors cancontain endogenous enhancers/promoters, splice junctions, interveningsequences, polyadenylation signals, etc. or a combination of bothendogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule. In some embodiments, recombinantnucleic acids are in an expression vector (e.g., plasmid), optionallyjoined to nucleic acids useful for driving expression of the nucleicacid (e.g., promoter or enhancer sequences).

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “5′-A-G-T-3′,” is complementary to thesequence “3′-T-C-A-S′.” Complementarity may be “partial,” in which onlysome of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “wild-type” when made in reference to a gene refers to a genethat has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring. A wild-type gene is frequently that gene which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a gene or to agene product refers, respectively, to a gene or to a gene product whichdisplays modifications in sequence and/or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

The term “isolated” when used in relation to a nucleic acid orpolypeptide, refers to a nucleic acid or polypeptide that issubstantially free of other proteins or nucleic acids (e.g., suitablefor pharmaceutical administration).

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used. Specificillustrative embodiments are described in the following.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments may include, but are not limited to,test tubes and cell cultures. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionsthat occur within a natural environment.

As used herein, “inhibitor” refers to a molecule which eliminates,minimizes, or decreases the activity, e.g., the biological, enzymatic,chemical, or immunological activity, of a target.

As used herein the term “disease” refers to a deviation from thecondition regarded as normal or average for members of a species, andwhich is detrimental to an affected individual under conditions that arenot inimical to the majority of individuals of that species (e.g.,diarrhea, nausea, fever, pain, inflammation, etc.).

As used herein, the term “administration” refers to the act of giving adrug, prodrug, antibody, or other agent, or therapeutic treatment to aphysiological system (e.g., a subject or in vivo, in vitro, or ex vivocells, tissues, and organs). Exemplary routes of administration to thehuman body can be through the eyes (ophthalmic), mouth (oral), skin(transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal),ear, by injection (e.g., intravenously, subcutaneously, intratumorally,intraperitoneally, etc.) and the like. “Coadministration” refers toadministration of more than one chemical agent or therapeutic treatment(e.g., radiation therapy) to a physiological system (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). As usedherein, administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order. “Coadministration” of therapeutictreatments may be concurrent, or in any temporal order or physicalcombination.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect, sign, or symptom of a disease or disorderthrough introducing in any way a therapeutic composition of the presenttechnology into or onto the body of a subject. “Treatment” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) the targetedpathologic condition or disorder. Those in need of treatment includethose already with the disorder as well as those prone to have thedisorder or those in whom the disorder is to be prevented.

As used herein, “therapeutically effective dose” refers to an amount ofa therapeutic agent sufficient to bring about a beneficial or desiredclinical effect. Said dose can be administered in one or moreadministrations. However, the precise determination of what would beconsidered an effective dose may be based on factors individual to eachpatient, including, but not limited to, the patient's age, size, type orextent of disease, stage of the disease, route of administration, thetype or extent of supplemental therapy used, ongoing disease process,and type of treatment desired (e.g., aggressive vs. conventionaltreatment).

As used herein, the term “effective amount” refers to the amount of acomposition sufficient to effect beneficial or desired results. Aneffective amount can be administered in one or more administrations,applications, or dosages and is not intended to be limited to aparticular formulation or administration route.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with, as desired, a carrier, inert oractive, making the composition especially suitable for diagnostic ortherapeutic use in vitro, in vivo, or ex vivo.

As used herein, the terms “pharmaceutically acceptable” or“pharmacologically acceptable” refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, “carriers” include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH-bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactants.

As used herein, the terms “patient” or “subject” refer to organisms tobe treated by the compositions of the present technology or to besubject to various tests provided by the technology. The term “subject”includes animals, preferably mammals, including humans. In a preferredembodiment, the subject is a primate. In an even more preferredembodiment, the subject is a human. In some embodiments, the subject isa companion animal (e.g., dog, cats, etc.), an agricultural animal(e.g., cow, sheep, goat, pig, etc.), or an equine.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to animal cells or tissues. In another sense, it ismeant to include a specimen or culture obtained from any source, such asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present technology.

Embodiments of the Technology

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

1. Inhibitors of SCF

Stem cell factor (SCF) is a ligand that is specific for the c-Kitreceptor kinase. Binding of SCF to c-Kit causes dimerization of c-Kitand activation of its kinase activity, which is important forhemopoiesis, melanogenesis, and fertility. Through c-Kit, SCF acts topromote cell survival, proliferation, differentiation, adhesion, andfunctional activation. Aberrant activation of c-Kit can result indisease, including fibrosis and tissue remodeling defects. Inparticular, there are multiple pulmonary diseases with known remodelingdefects as well as other chronic tissue remodeling diseases affectingother organs and tissues. Specific examples of diseases involvingfibrosis or tissue remodeling defects are idiopathic pulmonary fibrosis,chronic obstructive pulmonary disease, pulmonary arterial hypertension(PAH), asthma, acute respiratory distress syndrome, cystic fibrosis,peribronchial fibrosis, hypersensitivity pneumonitis, asthma,sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymalfibrosis, endomyocardial fibrosis, mediatinal fibrosis, nodularsubepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepaticfibrosis, fibromyalgia, gingival fibrosis, and radiation-inducedfibrosis.

Accordingly, interfering with the interaction between SCF and c-Kit canbe used to treat or study diseases involving aberrant activation ofc-Kit that causes fibrosis and tissue remodeling defects. The c-Kitreceptor is found on hematopoietic progenitor cells, melanocytes, germcells, eosinophils, lymphocytes, and mast cells. Thus, preventing SCFinteraction with c-Kit can alter the activation of severaldisease-associated cell populations that have been implicated infibrosis and tissue remodeling disease phenotypes.

Additionally, SCF induces key mediators in the fibrotic response, IL-25and IL-13. Data suggest that IL-25 can drive IL-13 expression in aT-cell and antigen-independent manner. Therefore, these processes canprogress without an antigen-specific response and consequentlychronically perpetuate remodeling and fibrotic disease. It iscontemplated that a complex cascade is established in which SCF inducesIL-25, which in turn induces production of IL-13, myofibroblastdifferentiation, and collagen production. IL-4 has also been identifiedas a fibrosis-associated cytokine.

2. Antibodies

In some embodiments, inhibiting the ability of SCF to interact withc-Kit is accomplished by means of an antibody that recognizes SCF. Insome embodiments, the antibody is a recombinant antibody (See e.g.,Example 1).

It is contemplated that antibodies against SCF find use in theexperimental, diagnostic, and therapeutic methods described herein. Incertain embodiments, the antibodies provided herein are used to detectthe expression of SCF in biological samples. For example, a samplecomprising a tissue biopsy can be sectioned and protein detected using,for example, immunofluorescence or immunohistochemistry. Alternatively,individual cells from a sample can be isolated, and protein expressiondetected on fixed or live cells by FACS analysis. Furthermore, theantibodies can be used on protein arrays to detect expression of SCF. Inother embodiments, the antibodies provided herein are used to decreasethe activity of cells expressing c-Kit by inhibiting SCF either in an invitro cell-based assay or in an in vivo animal model. In someembodiments, antibodies are used to treat a subject (e.g., humanpatient) by administering a therapeutically effective amount of anantibody against SCF.

In some embodiments, the present disclosure provides antibodies havingthe sequences of SEQ ID NOs:1-4 and variants thereof. For example, insome embodiments, the light chain variable domain comprises the aminoacid sequence of SEQ ID NO:4 or sequences with at least 80% (e.g., 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or fractions thereof) identity to SEQ ID NO:4; andthe heavy chain variable domain comprises the amino acid sequence of SEQID NO:2 or sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orfractions thereof identity to SEQ ID NO:2. In some embodiments, thelight chain variable region is encoded by the nucleic acid of SEQ IDNO:3 or sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orfractions thereof) homology to SEQ ID NO:3. In some embodiments, theheavy chain variable region is encoded by the nucleic acid of SEQ IDNO:1 or sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orfractions thereof) homology to SEQ ID NO:1.

In certain embodiments, antibodies are engineered, for example byincluding modifications of the Fc region which can alter serumhalf-life, complement fixation, Fc receptor binding and/or antigendependent cellular cytotoxicity.

In some embodiments, modified or variant antibodies as described hereinretain binding affinity to SCF (e.g., within 10%, 5%, 4%, 3%, 2%, or 1%)of the unmodified antibody. In some embodiments, binding affinity isincreased or decreased relative to the wild-type or unmodified antibody.

In certain embodiments, modifications in the biological properties of anantibody (e.g., those described herein) are accomplished by selectingsubstitutions that affect (a) the structure of the polypeptide backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget. The polynucleotide encoding a monoclonal antibody can further bemodified in a site, or (C) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (A. L. Lehninger, in Biochemistry, 2nd Ed., 73-75, WorthPublishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L),Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly(G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic:Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively,naturally occurring residues may be divided into groups based on commonside-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp,Glu; (4) basic: His, Lys, Arg; (5) residues that influence chainorientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class. Such substituted residues also may be introduced intothe conservative substitution sites or, into the remaining (e.g.,non-conserved) sites in a number of different manners using recombinantDNA.

In some embodiments, antibodies or antibody fragments are provided thatcan be produced which have altered glycosylation patterns. In certainembodiments, an antibody is altered to increase or decrease the extentto which the antibody is glycosylated. Glycosylation of polypeptides istypically either N-linked or O-linked. N-linked refers to the attachmentof a carbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

In some embodiments, the antibodies are PEGylated by reacting theantibody with a polyethylene glycol (PEG) derivative. In certainembodiments, the antibody is defucosylated and therefore lacks fucoseresidues.

In some embodiments, humanized anti-SCF antibodies are generated. Forexample, also contemplated are chimeric mouse-human monoclonalantibodies, which are produced by recombinant DNA techniques known inthe art. For example, a gene encoding the constant region of a murine(or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine constantregion, and the equivalent portion of a gene encoding a human constantregion is substituted (see, e.g., Robinson et al., PCT/US86/02269;European Patent Application 184,187; European Patent Application171,496; European Patent Application 173,494; WO 86/01533; U.S. Pat. No.4,816,567; European Patent Application 125,023 (each of which is hereinincorporated by reference in its entirety); Better et al., Science,240:1041-1043 (1988); Liu et al., Proc. Nat. Acad. Sci. USA,84:3439-3443 (1987); Liu et al., J. Immunol., 139:3521-3526 (1987); Sunet al., Proc. Nat. Acad. Sci. USA, 84:214-218 (1987); Nishimura et al.,Canc. Res., 47:999-1005 (1987); Wood et al., Nature, 314:446-449 (1985);and Shaw et al., J. Natl. Cancer Inst., 80:1553-1559 (1988)).

The chimeric antibody can be further humanized by replacing sequences ofthe variable region that are not directly involved in antigen bindingwith equivalent sequences from human variable regions. General reviewsof humanized chimeric antibodies are provided by S. L. Morrison,Science, 229:1202-1207 (1985) and by Oi et al., Bio Techniques, 4:214(1986). Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulinvariable regions from at least one of a heavy or light chain. Sources ofsuch nucleic acid are well known to those skilled in the art. Therecombinant DNA encoding the chimeric antibody, or fragment thereof, canthen be cloned into an appropriate expression vector.

Suitable humanized antibodies can alternatively be produced by CDRsubstitution (see, e.g., U.S. Pat. No. 5,225,539; Jones et al., Nature,321:552-525 (1986); Verhoeyan et al., Science, 239:1534 (1988); andBeidler et al., J. Immunol., 141:4053 (1988)). All of the CDRs of aparticular human antibody may be replaced with at least a portion of anon-human CDR or only some of the CDRs may be replaced with non-humanCDRs. It is only necessary to replace the number of CDRs important forbinding of the humanized antibody to the Fc receptor.

An antibody can be humanized by any method that is capable of replacingat least a portion of a CDR of a human antibody with a CDR derived froma non-human antibody. The human CDRs may be replaced with non-human CDRsusing oligonucleotide site-directed mutagenesis.

A humanized antibody may comprise one or more human and/or humanconsensus non-hypervariable region (e.g., framework) sequences in itsheavy and/or light chain variable domain. In some embodiments, one ormore additional modifications are present within the human and/or humanconsensus non-hypervariable region sequences. In one embodiment, theheavy chain variable domain of an antibody comprises a human consensusframework sequence, which in one embodiment is the subgroup IIIconsensus framework sequence. In one embodiment, an antibody comprises avariant subgroup III consensus framework sequence modified at least oneamino acid position. In one embodiment, the light chain variable domainof an antibody comprises a human consensus framework sequence, which inone embodiment is the κI consensus framework sequence. In oneembodiment, an antibody comprises a variant κI consensus frameworksequenced modified at least one amino acid position.

As is known in the art, and as described in greater detail herein below,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below). Embodiments of the invention provide antibodiescomprising modifications in these hybrid hypervariable positions. In oneembodiment, an antibody comprises a human variant human subgroupconsensus framework sequence modified at one or more hybridhypervariable positions.

An antibody as described herein can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In one embodiment, an antibody of embodiments of thedisclosure comprises at least a portion (or all) of the frameworksequence of human κ light chain. In one embodiment, an antibody ofembodiments of the disclosure comprises at least a portion (or all) ofhuman kappa subgroup I framework consensus sequence.

In some embodiments, antibodies are humanized antibodies or humanantibodies. Humanized forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodycomprises substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicityand HAMA response (human anti-mouse antibody) when the antibody isintended for human therapeutic use. Reduction or elimination of a HAMAresponse is a significant aspect of clinical development of suitabletherapeutic agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst.(1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawleret al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. ResponseMod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al.,J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323;Junghans et al., Cancer Res. (1990), 50:1495. As described herein, insome embodiments, the invention provides antibodies that are humanized.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human V domainsequence which is closest to that of the rodent is identified and thehuman framework region (FR) within it accepted for the humanizedantibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987)). Another method uses a particular frameworkregion derived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework may beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993)).

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), preferably the acceptor humanframeworks are from, or derived from, a human consensus frameworksequence as such frameworks have been demonstrated to have minimal, orno, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, embodiments of the present inventioncontemplate that the acceptor sequence may comprise pre-existing aminoacid substitutions relative to the human immunoglobulin sequence orhuman consensus framework sequence. These pre-existing substitutions arepreferably minimal; usually four, three, two or one amino aciddifferences only relative to the human immunoglobulin sequence orconsensus framework sequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35B(H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

While “incorporation” of hypervariable region residues is discussedherein, it will be appreciated that this can be achieved in variousways, for example, nucleic acid encoding the desired amino acid sequencecan be generated by mutating nucleic acid encoding the mouse variabledomain sequence so that the framework residues thereof are changed toacceptor human framework residues, or by mutating nucleic acid encodingthe human variable domain sequence so that the hypervariable domainresidues are changed to non-human residues, or by synthesizing nucleicacid encoding the desired sequence, etc.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins which bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants which can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, S., Proteins, 8:309(1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology,3:205 (1991)). In a monovalent phagemid display system, the gene fusionis expressed at low levels and wild type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S.Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have beenprepared in a number of ways including by altering a single gene byinserting random DNA sequences or by cloning a family of related genes.Methods for displaying antibodies or antigen binding fragments usingphage(mid) display have been described in U.S. Pat. Nos. 5,750,373,5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The library is then screened for expression of antibodies or antigenbinding proteins with the desired characteristics.

Additional methods of preparing and screening libraries are described,for example, in Geyer et al., Methods Mol Biol. 2012; 901:11-32; and deMarco A. Crit Rev Biotechnol. 2013 March; 33(1):40-8; each of which isherein incorporated by reference in its entirety.

Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. For example, hypervariable region residues can be substitutedusing the Kunkel method. See, e.g., Kunkel et al., Methods Enzymol.154:367-382 (1987).

According to another method, antigen binding may be restored duringhumanization of antibodies through the selection of repairedhypervariable regions (See US20060122377). The method includesincorporating non-human hypervariable regions onto an acceptor frameworkand further introducing one or more amino acid substitutions in one ormore hypervariable regions without modifying the acceptor frameworksequence. Alternatively, the introduction of one or more amino acidsubstitutions may be accompanied by modifications in the acceptorframework sequence.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent accessible and highly diverse positions in ahypervariable region. As described above, a codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps are isolated and cloned usingstandard recombinant techniques. As is well-established in the art,binding affinity of a ligand to its receptor can be determined using anyof a variety of assays, and expressed in terms of a variety ofquantitative values. Accordingly, in one embodiment, the bindingaffinity is expressed as Kd values and reflects intrinsic bindingaffinity (e.g., with minimized avidity effects). Generally andpreferably, binding affinity is measured in vitro, whether in acell-free or cell-associated setting. As described in greater detailherein, fold difference in binding affinity can be quantified in termsof the ratio of the monovalent binding affinity value of a humanizedantibody (e.g., in Fab form) and the monovalent binding affinity valueof a reference/comparator antibody (e.g., in Fab form) (e.g., a murineantibody having donor hypervariable region sequences), wherein thebinding affinity values are determined under similar assay conditions.Thus, in one embodiment, the fold difference in binding affinity isdetermined as the ratio of the Kd values of the humanized antibody inFab form and said reference/comparator Fab antibody. For example, in oneembodiment, if an antibody has an affinity that is “3-fold lower” thanthe affinity of a reference antibody (M), then if the Kd value for A is3×, the Kd value of M would be 1×, and the ratio of Kd of A to Kd of Mwould be 3:1. Conversely, in one embodiment, if an antibody has anaffinity that is “3-fold greater” than the affinity of a referenceantibody (R), then if the Kd value for C is 1×, the Kd value of R wouldbe 3×, and the ratio of Kd of C to Kd of R would be 1:3. Any of a numberof assays known in the art, including those described herein, are usedto obtain binding affinity measurements, including, for example,Biacore, radioimmunoassay (RIA) and ELISA technology to generatealternative antibodies. In one embodiment, the constant domains of thelight and heavy chains of, for example, a mouse monoclonal antibody canbe substituted 1) for those regions of, for example, a human antibody togenerate a chimeric antibody or 2) for a non-immunoglobulin polypeptideto generate a fusion antibody. In other embodiments, the constantregions are truncated or removed to generate the desired antibodyfragment of a monoclonal antibody. Furthermore, site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

It may be desirable to modify the antibody with respect to effectorfunction, e.g., so as to enhance or decrease antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

DNA encoding the Fv clones can be combined with known DNA sequencesencoding heavy chain and/or light chain constant regions (e.g. theappropriate DNA sequences can be obtained from Kabat et al., supra) toform clones encoding full or partial length heavy and/or light chains.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

It is further useful that antibodies be humanized with retention of highbinding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Also contemplated are chimeric and humanized antibodies in whichspecific amino acids have been substituted, deleted, or added. Inparticular, preferred humanized antibodies have amino acid substitutionsin the framework region, such as to improve binding to the antigen. Forexample, in a humanized antibody having mouse CDRs, amino acids locatedin the human framework region can be replaced with the amino acidslocated at the corresponding positions in the mouse antibody. Suchsubstitutions are known to improve binding of humanized antibodies tothe antigen in some instances.

In certain embodiments provided herein, it is desirable to use anantibody fragment. Various techniques are known for the production ofantibody fragments. Traditionally, these fragments are derived viaproteolytic digestion of intact antibodies (for example Morimoto et al.,1993, Journal of Biochemical and Biophysical Methods 24:107-117 andBrennan et al., 1985, Science, 229:81). For example, papain digestion ofantibodies produces two identical antigen-binding fragments, called Fabfragments, each with a single antigen-binding site, and a residual Fcfragment. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.

However, these fragments are now typically produced directly byrecombinant host cells as described above. Thus Fab, Fv, and scFvantibody fragments can all be expressed in and secreted from E. coli orother host cells, thus allowing the production of large amounts of thesefragments. Alternatively, such antibody fragments can be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

Fv is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy-chain and one light-chain variable domain in tight,non-covalent association. It is in this configuration that the threeCDRs of each variable domain interact to define an antigen-binding siteon the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known to the skilled artisan.

The technology herein provided also contemplates modifying an antibodyto increase its serum half-life. This can be achieved, for example, byincorporating a salvage receptor binding epitope into the antibodyfragment by mutation of the appropriate region in the antibody fragmentor by incorporating the epitope into a peptide tag that is then fused tothe antibody fragment at either end or in the middle (e.g., by DNA orpeptide synthesis).

The technology embraces variants and equivalents which are substantiallyhomologous to the chimeric, humanized, and human antibodies, or antibodyfragments thereof, provided herein. These can contain, for example,conservative substitution mutations, i.e. the substitution of one ormore amino acids by similar amino acids. For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class such as, for example, one acidic aminoacid with another acidic amino acid, one basic amino acid with anotherbasic amino acid, or one neutral amino acid by another neutral aminoacid. What is intended by a conservative amino acid substitution is wellknown in the art.

An additional embodiment utilizes the techniques known in the art forthe construction of Fab expression libraries (Huse et al., Science,246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Also, this technology encompasses bispecific antibodies thatspecifically recognize SCF. Bispecific antibodies are antibodies thatare capable of specifically recognizing and binding at least twodifferent epitopes. Bispecific antibodies can be intact antibodies orantibody fragments. Techniques for making bispecific antibodies arecommon in the art (Millstein et al., 1983, Nature 305:537-539; Brennanet al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol.121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al.,1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol.148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Pat.No. 5,731,168).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778; herein incorporated by reference) can be adapted toproduce specific single chain antibodies as desired. Single-chain Fvantibody fragments comprise the V_(H) and V_(L) domains of an antibody,wherein these domains are present in a single polypeptide chain.Preferably, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the single-chain Fvantibody fragments to form the desired structure for antigen binding.For a review of single-chain Fv antibody fragments, see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

Antibodies with the desired properties can be generated and purifiedusing any suitable method. In some embodiments, the expressedpolypeptides are secreted into and recovered from the periplasm of thehost cells. Protein recovery typically involves disrupting themicroorganism, generally by such means as osmotic shock, sonication orlysis. Once cells are disrupted, cell debris or whole cells may beremoved by centrifugation or filtration. The proteins may be furtherpurified, for example, by affinity resin chromatography.

Alternatively, proteins can be transported into the culture media andisolated therein. Cells may be removed from the culture and the culturesupernatant being filtered and concentrated for further purification ofthe proteins produced. The expressed polypeptides can be furtherisolated and identified using commonly known methods such aspolyacrylamide gel electrophoresis (PAGE) and Western blot assay.

In some embodiments, antibody production is conducted in large quantityby a fermentation process. Various large-scale fed-batch fermentationprocedures are available for production of recombinant proteins.Large-scale fermentations have at least 1000 liters of capacity,preferably about 1,000 to 100,000 liters of capacity. These fermentorsuse agitator impellers to distribute oxygen and nutrients, especiallyglucose (the preferred carbon/energy source). Small scale fermentationrefers generally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters. Medium scale reactors are reactors of100 L-1000 L.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the antibodies, variousfermentation conditions can be modified. For example, to improve theproper assembly and folding of the secreted antibody polypeptides,additional vectors overexpressing chaperone proteins, such as Dsbproteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolylcis,trans-isomerase with chaperone activity) can be used to co-transformthe host prokaryotic cells. The chaperone proteins have beendemonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereofome E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system.

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

2. Therapies Using Inhibitors of SCF

Inhibiting SCF finds use in therapies to treat disease. Accordingly,provided herein are therapies comprising inhibiting SCF to benefitindividuals suffering from disease. In particular, as shown herein,disease states involving fibrosis and tissue remodeling demonstrateaberrant SCF activity. For example, fibroblasts isolated from diseasedindividuals with fibrotic or tissue remodeling phenotypes directlyrespond to SCF, which results in the generation of a more severephenotype that includes increased collagen production. As such, as shownherein, inhibiting SCF can significantly affect the generation of severedisease consequences including inflammation and remodeling of targettissue. Also contemplated are therapies targeting SCF during thegeneration of fibrosis associated with acute and chronic disorders thathave either a dynamic disease course or a more predictable diseasecourse. Indications that can benefit from therapy inhibiting SCFinclude, but are not limited to, idiopathic pulmonary fibrosis,pulmonary arterial hypertension (PAH), chronic obstructive pulmonarydisease, acute respiratory distress syndrome, cystic fibrosis,peribronchial fibrosis, hypersensitivity pneumonitis, asthma,sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymalfibrosis, endomyocardial fibrosis, mediatinal fibrosis, nodularsubepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepaticfibrosis, fibromyalgia, gingival fibrosis, and radiation-inducedfibrosis.

Importantly, therapies targeting SCF reduce or eliminate toxic effectsassociated with other similar therapies, for example those targetingc-Kit. These undesirable toxic effects are associated with targeting anintracellular, rather than extracellular, target, and the morewidespread and general changes in cell signaling that result. While thetherapies are not limited in their route of administration, embodimentsof the technology provided herein deliver the SCF inhibitor via theairway by intranasal administration. Such administration allows directdelivery of the therapeutic agent to target tissues in pulmonarydiseases involving fibrosis and tissue remodeling, rather than relyingon systemic delivery via an orally administered composition.

In certain embodiments, a physiologically appropriate solutioncontaining an effective concentration of an antibody specific for SCFcan be administered topically, intraocularly, parenterally, orally,intranasally, intravenously, intramuscularly, subcutaneously, or by anyother effective means. In particular, the antibody may delivered into anairway of a subject by intranasal administration. Alternatively, atissue can receive a physiologically appropriate composition (e.g., asolution such as a saline or phosphate buffer, a suspension, or anemulsion, which is sterile) containing an effective concentration of anantibody specific for SCF via direct injection with a needle or via acatheter or other delivery tube. Any effective imaging device such asX-ray, sonogram, or fiber-optic visualization system may be used tolocate the target tissue and guide the administration. In anotheralternative, a physiologically appropriate solution containing aneffective concentration of an antibody specific for SCF can beadministered systemically into the blood circulation to treat tissuethat cannot be directly reached or anatomically isolated. Suchmanipulations have in common the goal of placing an effectiveconcentration of an antibody specific for SCF in sufficient contact withthe target tissue to permit the antibody specific for SCF to contact thetissue.

With respect to administration of a SCF inhibitor (e.g., an antibodyspecific for SCF) to a subject, it is contemplated that the SCFinhibitor be administered in a pharmaceutically effective amount. One ofordinary skill recognizes that a pharmaceutically effective amountvaries depending on the therapeutic agent used, the subject's age,condition, and sex, and on the extent of the disease in the subject.Generally, the dosage should not be so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema, congestiveheart failure, and the like. The dosage can also be adjusted by theindividual physician or veterinarian to achieve the desired therapeuticgoal.

As used herein, the actual amount encompassed by the term“pharmaceutically effective amount” will depend on the route ofadministration, the type of subject being treated, and the physicalcharacteristics of the specific subject under consideration. Thesefactors and their relationship to determining this amount are well knownto skilled practitioners in the medical, veterinary, and other relatedarts. This amount and the method of administration can be tailored toachieve optimal efficacy but will depend on such factors as weight,diet, concurrent medication, and other factors that those skilled in theart will recognize.

In some embodiments, a SCF inhibitor (e.g., an antibody specific forSCF) according to the technology provided herein is administered in apharmaceutically effective amount. In some embodiments, a SCF inhibitor(e.g., an antibody specific for SCF) is administered in atherapeutically effective dose. The dosage amount and frequency areselected to create an effective level of the SCF inhibitor withoutsubstantially harmful effects. When administered, the dosage of a SCFinhibitor (e.g., an antibody specific for SCF) will generally range from0.001 to 10,000 mg/kg/day or dose (e.g., 0.01 to 1000 mg/kg/day or dose;0.1 to 100 mg/kg/day or dose).

Pharmaceutical compositions preferably comprise one or more antibodiesas described herein associated with one or more pharmaceuticallyacceptable carriers, diluents, or excipients. Pharmaceuticallyacceptable carriers are known in the art such as those described in, forexample, Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R.Gennaro ed., 1985).

For administration by inhalation, the compounds are delivered in theform of an aerosol spray or dry powder from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer. Compositions for inhalation orinsufflation include solutions and suspensions in pharmaceuticallyacceptable, aqueous or organic solvents, or mixtures thereof, andpowders. The liquid or solid compositions may contain suitablepharmaceutically acceptable excipients as described supra. In someembodiments, the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions in can benebulized by use of inert gases. Nebulized solutions may be breatheddirectly from the nebulizing device or the nebulizing device can beattached to a face masks tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder compositions can beadministered orally or nasally from devices which deliver theformulation in an appropriate manner.

In some embodiments, a single dose of a SCF inhibitor (e.g., an antibodyspecific for SCF) according to the technology provided herein isadministered to a subject. In other embodiments, multiple doses areadministered over two or more time points, separated by hours, days,weeks, etc. In some embodiments, compounds are administered over a longperiod of time (e.g., chronically), for example, for a period of monthsor years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months oryears; e.g., for the lifetime of the subject). In such embodiments,compounds may be taken on a regular scheduled basis (e.g., daily,weekly, etc.) for the duration of the extended period.

In some embodiments, a SCF inhibitor (e.g., an antibody specific forSCF) according to the technology provided herein is co-administered withanother compound or more than one other compound (e.g., 2 or 3 or moreother compounds). Examples include, but are not limited to,immunosuppressive therapy such as corticosteroids andimmunosuppressants, such as cyclophosphamide, azathioprine,methotrexate, penicillamine, and cyclosporine.

3. Kits

Some embodiments provide herein kits for the treatment of a subject. Insome embodiments, the kits include an antibody that binds to SCF andappropriate solutions and buffers. Embodiments include all controls andinstructions for use. In some embodiments, kits include delivery systems(e.g., injectors, inhalers, nebulizers, etc.).

EXAMPLES Example 1 Isolation and Sequencing of Monoclonal Antibodies toSCF

A. Methods Total RNA was extracted from fresh hybridoma cells recoveredby GenScript and cDNA was synthesized from the RNA. RT-PCR was thenperformed to amplify the variable regions (heavy and light chains) ofthe antibody, which were then cloned into a standard cloning vectorseparately and sequenced.

Hybridoma cells recovered by GenScript; TRIzol® Plus RNA PurificationSystem (Invitrogen, Cat. No.: 15596-026); SuperScript™ III First-StrandSynthesis System (Invitrogen, Cat. No.: 18080-051).

Total RNA was isolated from the hybridoma cells following the technicalmanual of TRIzol® Plus RNA Purification System. The total RNA wasanalyzed by agarose gel electrophoresis.

Total RNA was reverse transcribed into cDNA using isotype-specificanti-sense primers or universal primers following the technical manualof SuperScript™ III First-Strand Synthesis System. The antibodyfragments of VH and VL were amplified according to the standardoperating procedure of RACE of GenScript.

Amplified antibody fragments were separately cloned into a standardcloning vector using standard molecular cloning procedures.

Colony PCR screening was performed to identify clones with inserts ofcorrect sizes. No less than five single colonies with inserts of correctsizes were sequenced for each antibody fragment.

B. Results

Five single colonies with correct VH and VL insert sizes were sequenced.The VH and VL genes of five different clones were found nearlyidentical. The consensus sequence, listed below, is the sequence of theantibody produced by the hybridoma 2G8D3.

Heavy chain: DNA sequence (402 bp; SEQ ID NO:1)

Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCTTATCCCAAGTTTCTCTAAAAGAGTCTGGCCCTGGGATATTGAGGCCCTCACAGACCCTCATTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGTACTTCTGGTATGGGTGTGGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGAGAAGTCCTATAACCCATCCCTGAAGAGCCGGCTCACGATCTCCAAGGATGCCTCCCGAGACCAGGTTTTCCTCAAGATCACCAATGTGGACACTACAGATACTGCCACTTACTTCTGTGCTCGAAGCGGCTTGGACTACTGGGGTCAAGGAATT TCAGTCACCGTCTCCTCA

Heavy chain: Amino acids sequence (134 AA; SEQ ID NO:2)

Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

MDRLTSSFLLLIVPAYVLSQVSLKESGPGILRPSQTLILTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDEKSYNPSLKSRLTISKDASRDQVFLKITNVDTTDTATYFCARSGLDYWGQGISVTVSS

Light chain: DNA sequence (402 bp; SEQ ID NO:3)

Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

ATGGCCTGGACTCCTCTCTTCTTCTTCTTTGTTCTTCATTGCTCAGGTTCTTTCTCCCAACCTGTGCTCACTCAGTCATCTTCAGCCTCTTTCTCCCTGGGAGCCTCAGCAAAAATCACGTGCACCTTGAGTAGTCAGCACAGGACGTACACCATTGAATGGTATCAGCAACAGCCACTCAAGCCTCCTAAGTATGTGATGGAACTTAAGAGAGATGGAAGTCACAGAACAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTCTGGTGCTGATCGCTACCTAACCATTGCCAACATCCAGCCTGAAGATGAAGCAATGTACATCTGTGGTGCTGATGATACAATTCAGGAACAATTTGTGTATGTTTTCGGCGGTGGA ACCAAAGTCACTGTCCTC

Light chain: Amino acids sequence (134 AA; SEQ ID NO:4)

Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

MAWTPLFFFFVLHCSGSFSQPVLTQSSSASFSLGASAKITCTLSSQHRTYTIEWYQQQPLKPPKYVMELKRDGSHRTGDGIPDRFSGSSSGADRYLTIANIQPEDEAMYICGADDTIQEQFVYVFGGGTKVTVL

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inpharmacology, biochemistry, medical science, or related fields areintended to be within the scope of the following claims.

We claim:
 1. An isolated recombinant monoclonal anti-stem cell factor(SCF) antibody comprising: (a) a light chain variable domain comprisingthe amino acid sequence of SEQ ID NO:4 or sequences with at least 80%identity to SEQ ID NO:4; and (b) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:2 or sequences with atleast 80% identity to SEQ ID NO:2.
 2. The antibody of claim 1, whereinsaid light chain variable region comprises the amino acid sequence ofSEQ ID NO:4 or sequences with at least 85% identity to SEQ ID NO:4 andsaid heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:2 or sequences with at least 85% identity to SEQ ID NO:2. 3.The antibody of claim 1, wherein said light chain variable regioncomprises the amino acid sequence of SEQ ID NO:4 or sequences with atleast 90% identity to SEQ ID NO:4 and said heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:2 or sequences with atleast 90% identity to SEQ ID NO:2.
 4. The antibody of claim 1, whereinsaid light chain variable region comprises the amino acid sequence ofSEQ ID NO:4 or sequences with at least 95% identity to SEQ ID NO:4 andsaid heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:2 or sequences with at least 95% identity to SEQ ID NO:2. 5.The antibody of claim 1, wherein said light chain variable regioncomprises the amino acid sequence of SEQ ID NO:4 or sequences with atleast 98% identity to SEQ ID NO:4 and said heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:2 or sequences with atleast 98% identity to SEQ ID NO:2.
 6. The antibody of claim 1, whereinsaid light chain variable region has the amino acid sequence of SEQ IDNO:4 and heavy chain variable region has the amino acid sequence of SEQID NO:2.
 7. The antibody of claim 1, wherein the antibody is monovalentand comprises an Fc region.
 8. The antibody of claim 1, wherein theantibody is bivalent.
 9. The antibody of claim 1, wherein the antibodyis bispecific.
 10. The antibody of claim 1, wherein the antibody is anantibody fragment selected from the group consisting of a Fab, aFab′-SH, an Fv, an scFv, and a (Fab′).sub.2 fragment.
 11. The antibodyof claim 1, wherein the antibody comprises a single Fab region linked toan Fc region.
 12. A nucleic acid encoding the antibody of claim
 1. 13.The nucleic acid of claim 12, comprising a nucleic acid encoding a lightchain variable region comprising SEQ ID NO:3 or sequences that are atleast 80% homologous to SEQ ID NO:3 and a nucleic acid encoding a heavychain variable region comprising SEQ ID NO:1 or sequences that are atleast 80% homologous to SEQ ID NO:1.
 14. The nucleic acid of claim 13,wherein said nucleic acid encoding a light chain variable regioncomprises SEQ ID NO:3 or sequences that are at least 85% homologous toSEQ ID NO:3 and said nucleic acid encoding a heavy chain variable regioncomprises SEQ ID NO:1 or sequences that are at least 85% homologous toSEQ ID NO:1.
 15. The nucleic acid of claim 13, wherein said nucleic acidencoding a light chain variable region comprises SEQ ID NO:3 orsequences that are at least 90% homologous to SEQ ID NO:3 and saidnucleic acid encoding a heavy chain variable region comprises SEQ IDNO:1 or sequences that are at least 90% homologous to SEQ ID NO:1. 16.The nucleic acid of claim 13, wherein said nucleic acid encoding a lightchain variable region comprises SEQ ID NO:3 or sequences that are atleast 95% homologous to SEQ ID NO:3 and said nucleic acid encoding aheavy chain variable region comprises SEQ ID NO:1 or sequences that areat least 95% homologous to SEQ ID NO:1.
 17. The nucleic acid of claim13, wherein said nucleic acid encoding a light chain variable regioncomprises SEQ ID NO:3 or sequences that are at least 98% homologous toSEQ ID NO:3 and said nucleic acid encoding a heavy chain variable regioncomprises SEQ ID NO:1 or sequences that are at least 98% homologous toSEQ ID NO:1.
 18. The nucleic acid of claim 13, wherein said nucleic acidencoding a light chain variable region comprises SEQ ID NO:3 and saidnucleic acid encoding a heavy chain variable region comprises SEQ IDNO:1.
 19. A method of treating a fibrotic or tissue remodeling diseasecomprising administering a therapeutically effective amount of thepharmaceutical composition of claim 12 to a subject with or at risk fora fibrotic or tissue remodeling disease.
 20. The method of claim 19,wherein the disease is fibrosis, a remodeling disease, or a pulmonarydisease.